Since decades, expression vectors have been used as vehicles for the expression of genes or cDNAs encoding polypeptides or proteins of interest in host cells. Strong viral or cellular promoters and enhancers are being used to express the gene of interest at high levels by using transient or stable transfection of recombinant DNA into the host cells. The immediate early (IE) region of the human cytomegalovirus (hCMV) has been shown to be particularly suitable in this regard, and expression vectors comprising gene elements derived from this region are known e.g. from EP0323997B1.
Until today, the gene regulatory sequences from the murine cytomegalovirus (mCMV) have been used rarely, although mCMV derived regulatory elements were identified to be very powerful and even stronger than the human counterpart (Kim et al. 2002).
U.S. Pat. No. 4,963,481 (de Villiers) discloses expression vectors having a DNA encoding a heterologous protein under the transcriptional control of DNA fragments derived from the mCMV IE gene region including an approximately 2270 base pair (bp) restriction endonuclease PstI fragment isolated from the viral genome. A 1387 bp truncated version of this fragment resulted in significant improvement in the efficacy of the DNA fragment as a promoter for expression of the heterologous protein.
U.S. Pat. No. 4,968,615 (Kozinowski) describes recombinant DNA molecules containing transcription enhancers from murine cytomegalovirus (mCMV) which can be used to enhance the transcription of structural genes in eukaryotic cells. The mCMV enhancer was said to be located within the 2.27 kb PstU fragment identified by de Villiers (U.S. Pat. No. 4,963,481).
Manning and Mocarski (1988) analysed the functional importance of the mCMV IE2 region for replication of the murine cytomegalovirus. To this end, a recombinant virus was constructed having the lacZ reporter gene under the transcriptional control of the mCMV IE enhancer/promoter, thus disrupting the IE2 gene. Indeed no IE2 gene expression could be observed, and the virus replicated normally. The authors thus concluded that the IE2 gene, that is not conserved among the cytomegaloviruses such as the mCMV and the hCMV, was not essential for virus replication. No indication of any particular utility of the IE2 enhancer/promoter region was disclosed or suggested by Manning and Mocarski.
More recent literature shows a further difference between the mouse and human CMV IE region. The mouse locus expresses a second major mRNA in the opposite direction of the first IE gene. This second immediate early gene was termed IE2, and its promoter sequence referred to as IE2 promoter (Messerle et al. 1991).
The IE2 region from the mCMV has not been used in vectors for expression of heterologous proteins so far.